You’d be accurate in describing the difficulties of protein identification by pointing out that “blood is thicker than water”. While this idiom usually refers to the strong bond of family relationships, the fact is that blood is indeed thick with its complex composition of different proteins and other components. Life depends on such diversity. However, teasing those components apart for individual study has challenged generations of researchers. Today’s products provide the best strategies to date by melding new technologies with the decades of experience.
Blood has long proved useful for assessing the body’s health. Along with the ease of obtaining samples, blood contains a collection of proteins that represents all those present in the body. But researchers are now starting to reconsider the convenience of blood with the realization that important proteins “could be present at extremely low concentrations” according to a paper published this year in the journal
Molecular & Cellular Proteomics. Thus, raising “doubts as to whether disease biomarkers can be accurately detected or identified from plasma using a proteomics approach.” (WJ Qian et al, “Challenges in Biomarker Discovery: Advances and Challenges in Liquid Chromatography-Mass Spectrometry-based Proteomics Profiling for Clinical Applications”, Molecular & Cellular Proteomics, 5:1727-1744, 2006.) Such difficulties have led researchers to consider other bodily fluids, such as cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, saliva, urine, and tumor interstitial fluids.
Whichever serum or slime you’re analyzing, low abundant proteins in blood may go undetected because they are “dominated by several very abundant proteins (i.e. the 22 most abundant proteins represent 99% of the total protein mass in plasma),” according to the paper. “Yet at the same time [the collection of proteins in the plasma] presents an extraordinary dynamic range (>10 orders of magnitude) in protein concentrations that begins with serum albumin at 45 mg/ml and extends to cytokines (and potentially many disease-related proteins) at around 1–10 pg/ml or lower.” Add in the variability of post-translational modifications, splice variants, and human genetic heterogeneity, and the difficulties of protein isolation become apparent.
Most proteomic experts would agree that they lack a technology that can circumvent every problem of protein purification. Their general consensus is that new technologies have allowed them to make leaps and bounds, but not completely reach the finishing line. The current belief is that you can obtain optimal results with the synergy of several different fractionation and purification techniques. The market now offers the tools and supplies for this multi-pronged strategy. The latest products are more convenient with kits specifically designed for certain types of proteins, samples or other upstream and downstream techniques you may perform.
Magnetic beads, which have gained immense popularity in the last several years, are well represented among the most efficient protocols. You can use beads to remove unwanted salts and contaminants, concentrate and fractionate proteins, and remove abundant proteins. To concentrate tagged proteins, purchase beads with the appropriate ligands. For other types of proteins, purchase beads with specific antibodies. To retrieve the beads, simply apply a magnetic field. For those who favor spin columns, the market offers plenty of kits to suit your fancy. While some spin columns are made for removing detergents from your protein sample, other spin chromatography products can increase the concentration of proteins of particular molecular weights.
Take a look at the products below to get started with wending your way through the immense world of proteins.
Detergents are extensively used to prepare protein samples. However, detergents must often be removed prior to downstream analysis because of their undesirable effects. These include extraneous peaks in mass spectrometry, artifacts with chromatography and electrophoresis, interference with microinjection into cells and interference with protein immunization. The ProteoSpin™ Detergent Clean-up Micro Kit provides a fast and simple procedure for the effective removal of SDS, Triton® X-100 and other detergents from small samples of protein. The kit employs spin-column chromatography using Norgen’s patented protein resin as an ion exchanger.
The new ProteoExtract® Formalin Fixed Tissue Kit utilizes proprietary detergent-free reagents to facilitate the extraction of full-length, soluble proteins directly from formalin-fixed (FF) and paraffin embedded
(FFPE) tissue sections as well as from various types of tissues (species, organs, normal and diseased). Following extraction, the samples are compatible with downstream applications such as separation through gel electrophoresis followed by Western blot analysis, tryptic digestion, and mass spectrometry. Each kit contains sufficient reagents to process 10–30 mg tissue samples of formalin-fixed tissue.
